GENETICALLY ENGINEERED SENSORS FOR IN VIVO DETECTION OF BLEEDING

• US 20170058282A1
• Filed: 07/08/2016
• Published: 03/02/2017
• Est. Priority Date: 07/09/2015
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Mimee, Mark K., Lu, Timothy Kuan-Ta
• Application Number:
  US15/205,625
• Publication Number:
  US 20170058282A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/74 Bacteria therapeutic use of a bacterial protein A61K38/00
• CPC Class Codes: A61K 49/0013 Luminescence
• CPC Class Codes: A61K 49/0017 Fluorescence in vivo
• CPC Class Codes: A61K 49/0069 the agent being in a particular physical galenical form
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)
• CPC Class Codes: C07K 14/335 from Lactobacillus (G)
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2800/22 Haematology

Abstract

Provided herein are microorganisms engineered with heme-responsive transcription factors and genetic circuits. Also provided are methods for using engineered microorganisms to sense bleeding events and treat bleeding in vivo.

Claims

1. An engineered microorganism comprising

a heme-responsive transcription factor;

and

a genetic circuit responsive to the heme-responsive transcription factor.

2. The engineered microorganism of claim 1 further comprising a heme transporter.

3. The engineered microorganism of claim 1, wherein the heme-responsive transcription factor is a TetR-family transcriptional repressor, and/or wherein the heme responsive transcription factor is from Lactococcus lactis, optionally wherein the heme responsive transcription factor is HrtR.

6. The engineered microorganism of claim 1, wherein the microorganism is Escherichia coli or Lactococcus lactis.

8. The engineered microorganism of claim 2, wherein the heme transporter is from a gram negative bacterium, and/or wherein the heme transporter is ChuA.

10. The engineered microorganism of claim 1, wherein the genetic circuit comprises a first promoter that is operably linked to a nucleic acid sequence encoding an output molecule, wherein the output molecule is a nucleic acid, a reporter polypeptide, a recombinase, or a therapeutic protein, and wherein the first promoter is responsive to the heme-responsive transcription factor.

11. The engineered microorganism of claim 10, wherein the first promoter is a P_HrtAB(HrtR) promoter or a P_L promoter with one or more HrtO operator sites.

12. The engineered microorganism of claim 1, further comprising a second promoter that is operably linked to a nucleic acid encoding the heme-responsive transcription factor.

13. The engineered microorganism of claim 12, wherein the nucleic acid encoding the heme-responsive transcription factor encodes a ribosomal binding site (RBS), wherein the nucleic acid encoding the RBS comprises the nucleic acid sequence SEQ ID NO:

17, SEQ ID NO;

18, SEQ ID NO;

19, or SEQ ID NO;

20.

14. The engineered microorganism of claim 1, further comprising a third promoter that is operably linked to a nucleic acid encoding the heme transporter.

17. The engineered microorganism of claim 1, wherein the heme-responsive transcription factor is a response regulator from a two-component system, optionally wherein the heme responsive transcription factor is HssR.

19. The engineered microorganism of claim 17, wherein the engineered microorganism further comprises a heme-responsive histidine kinase from a two-component system, optionally wherein the heme-responsive histidine kinase is HssS.

21. The engineered microorganism of claim 19, wherein the genetic circuit comprises

(a) a first promoter that is operably linked to a nucleic acid sequence encoding an output molecule, wherein the first promoter is responsive to the response regulator from a two-component system,

(b) optionally a second promoter that is operably linked to a nucleic acid encoding the response regulator from a two-component system, and

(c) optionally a third promoter that is operably linked to a nucleic acid encoding the heme-responsive histidine kinase from a two-component system.

29. The engineered microorganism of claim 1, comprising a first plasmid, wherein the first plasmid encodes the heme-responsive transcription factor, the genetic circuit responsive to the heme-responsive transcription factor, and/or a heme transporter.

30. The engineered microorganism of claim 29, further comprising a second plasmid, wherein the second plasmid encodes the heme-responsive transcription factor, the genetic circuit responsive to the heme-responsive transcription factor, and/or a heme transporter.

34. A method of detecting and/or treating bleeding in a subject comprising administering to the subject the engineered microorganism of claim 1.

39. The method of claim 34, wherein the subject has, or is at risk of having a bleeding disease or disorder selected from the group consisting of colitis, peptic ulcer disease, liver cirrhosis, inflammatory bowel disease, hemorrhoids, an infection, cancer, a vascular disorder, an adverse effect of a medication, and a blood clotting disorder.

43. The method of claim 34, further comprising obtaining the engineered microorganism from the subject and analyzing the engineered microorganism in vitro.

45. The method of claim 43, wherein analyzing the engineered microorganism comprises polymerase chain reaction (PCR), nucleic acid sequencing, measuring the level of an output molecule, measuring fluorescence or luminescence from the engineered microorganism, and/or measuring an amount of the engineered microorganism obtained from the subject.

46. The method of claim 34, further comprising analyzing the engineered microorganism in vivo.

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